Endotoxins are lipopolysaccharides (LPS) found in the outer membrane of gram-negative bacteria such as E. coli (Raetz, Ann. Rev. Biochem., 1990, p. 129, Vol. 59). The LPS molecule contains three distinct chemical regions, the lipid A region, a central polysaccharide region and the O-antigen region. The lipid A region is composed of a glucosamine disaccharide containing phosphate groups and is highly substituted with long chain fatty acids and is thought to be responsible for the toxic effects of endotoxins (Rietschel et al, Cellular and Molecular Aspects of Endotoxin Reaction, Ed. Nowotny, A., Spitzner, J. J. and Ziegler, E. J., 1990, p. 15).
The biological effects induced by endotoxins result from activation of the immune system, especially the monocytes and macrophages, and are extremely diverse including fever, metabolic breakdown and septic shock (Martich et al, Immunobiology, 1993, p. 403, Vol. 187). The presence of endotoxins in biologically derived products and pharmaceutical products for therapeutic use is an area of major concern due to the potentially harmful effects of endotoxins. Although maintaining sterile processes can ensure that biological products are free from endotoxins, this is not often possible especially when the biological product is expressed in a gram-negative bacterial source such as E. coli. Typical processes for inactivating endotoxins include exposure to concentrated sodium hydroxide (1M NaOH) and/or heat treatment (250° C.) for prolonged periods of time. However, such treatment processes are not applicable to the majority of biological products.
Numerous techniques have been used to remove endotoxins from biological products and include ultrafiltration (Sweadner et al, Appl. Environ. Microbiol., 1977, p. 382, Vol. 34; Li et al, Biotechnol. Tech., 1998, p. 119, Vol. 12), charcoal adsorption (Nagaki et al, Int. J. Artif. Organs., 1991, p. 43, Vol. 14), size exclusion chromatography, ion exchange chromatography (Hou et al, J. Parenter. Sci. Technol., 1990, p. 204, Vol. 44; Hou et al, Biotechnol. Bioeng., 1990, p. 315, Vol. 12; Neidhardt et al, J. Chromatogr., 1992, p. 255, Vol. 590) and affinity chromatography. All these techniques exhibit significant drawbacks and especially when applied to endotoxin removal from therapeutic proteins and other biological products. Charcoal adsorption and ion exchange chromatography reduce endotoxin levels from protein solutions, but they also tend to bind the biological component.
Efficient endotoxin removal has been achieved using affinity chromatography on immobilized polymixin B (Talmadge et al, J. Chromatogr., 1989, p. 175, Vol. 476; Anspach et al, J. Chromatogr. A, 1995, p. 81, Vol. 711). However, concerns over the potential toxicity of polymixin B leachates has limited its use. Synthetic ligands based on diaminoalkane and monoaminoalkane compounds attached to solid phase matrices have been used for the removal of endotoxins from aqueous solutions but have only shown a limited capacity for binding endotoxins (Hou et al, Biochim. Biophys. Acta, 1991, p. 149, Vol. 1073).
J. Chromatography 248, 401-408 (1982) and 262, 193-198 (1983), and also U.S. Pat. No. 4,381,239, describe histamine and other aromatic nitrogen heterocycles covalently linked to solid phase matrices.
U.S. Pat. No. 5,358,933 describes synthetic peptides for detoxification of bacterial endotoxin and for the prevention and treatment of septic shock.